CN117255945A

CN117255945A - Nanopore sensor device

Info

Publication number: CN117255945A
Application number: CN202280024381.XA
Authority: CN
Inventors: B·博扬诺瓦; J·G·曼德尔; S·M·麦克唐纳
Original assignee: Illumina Inc
Current assignee: Illumina Inc
Priority date: 2021-03-31
Filing date: 2022-03-01
Publication date: 2023-12-19
Also published as: AU2022246740A1; WO2022211998A1; EP4314810A1; EP4314811A1; CA3206267A1; JP2024511700A; KR20230165244A

Abstract

Examples of nanopore sensor devices include: one or more cis traps; a cis electrode; a plurality of trans-wells, each of the plurality of trans-wells separated from the one or more cis-wells by a lipid/polymer/solid state membrane having nanopores; a plurality of counter electrodes, each counter electrode of the plurality of counter electrodes being associated with one of the plurality of counter wells; a first concentration of an electrolyte, the first concentration of the electrolyte being located within the one or more cis wells; and a second concentration of the electrolyte, the second concentration of the electrolyte being located within the trans trap, wherein the first concentration is higher than the second concentration.

Description

Nanopore sensor device

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application Ser. No. 63/168,646 filed on 3/31 of 2021, the contents of which are incorporated herein by reference in their entirety.

Background

Various polynucleotide sequencing techniques involve performing a number of controlled reactions on a localized support surface or within a predefined reaction chamber. The designated reaction may then be observed or detected, and subsequent analysis may help identify or reveal the identity of the polynucleotides involved in the reaction. Another polynucleotide sequencing technique has been developed that utilizes nanopores that provide a channel for ionic current. The polynucleotide or label/tag that binds the nucleotide is driven into the nanopore, thereby changing the resistivity of the nanopore. Each nucleotide (or series of nucleotides) or each tag/small tag (or series of tags/tags) produces a characteristic electrical signal, and the record of signal levels corresponds to the sequence of the polynucleotide. In existing nanopore sensor devices (at t=0), the current is equally carried by the electrolyte that translocates through the nanopore in opposite directions between the cis and trans traps. However, such nanopore sequencing devices have a low lifetime.

Disclosure of Invention

In a first example, a nanopore sensor device includes: one or more cis traps; a cis electrode; a plurality of trans-wells, each of the plurality of trans-wells separated from the one or more cis-wells by a lipid/polymer/solid state membrane having nanopores; a plurality of counter electrodes, each counter electrode of the plurality of counter electrodes being associated with one of the plurality of counter wells; a first concentration of an electrolyte, the first concentration of the electrolyte being located within the one or more cis wells; and a second concentration of the electrolyte, the second concentration of the electrolyte being located within the trans trap, wherein the first concentration is higher than the second concentration.

In a second example, a nanopore sensor kit includes: i) A nanopore sensor device, the nanopore sensor device comprising: one or more cis traps, the one or more cis traps comprising a fluid inlet; a cis electrode; a plurality of trans-wells, each of the plurality of trans-wells separated from the one or more cis-wells by a lipid/polymer/solid state membrane having nanopores; a plurality of counter electrodes, each counter electrode of the plurality of counter electrodes being associated with one of the plurality of counter wells; and a first concentration of an electrolyte, the first concentration of the electrolyte being located within the one or more cis-wells and the plurality of trans-wells; and ii) a second concentration of the electrolyte to be introduced into the one or more cis-wells through the fluid inlet upon initial cycling of the nanopore sensor device such that the one or more cis-wells contain the second concentration of the electrolyte and the plurality of trans-wells contain the first concentration of the electrolyte, wherein the second concentration is higher than the first concentration.

In a third example, a method of detecting ion current to analyze biological compounds includes: providing a nanopore within a membrane separating a cis-well and a trans-well, the nanopore having a plurality of positively charged residues on an inner surface of the nanopore; providing an electrolyte within the cis well and the trans well; and applying a current between a cis cathode at least partially exposed to the cis well and a trans anode at least partially exposed to the trans well to generate an ion current through the nanopore, wherein the plurality of positively charged residues of the nanopore inhibit translocation of cations from the trans well to the cis well during application of the current.

In a fourth example, a nanopore sensor device includes: one or more cis traps; a cis electrode; a plurality of trans-wells, each of the plurality of trans-wells separated from the one or more cis-wells by a lipid/polymer/solid state membrane having nanopores; a plurality of counter electrodes, each counter electrode of the plurality of counter electrodes being associated with one of the plurality of counter wells; an electrolyte solution comprising a redox inactive buffer comprising an anion having a diameter greater than the diameter of the constriction region of the nanopore and a redox species.

It should be appreciated that any of the features of any of the examples set forth herein may be combined in any desired manner. For example, any combination of features of the first and/or second and/or third and/or fourth examples may be used together, and/or may be combined with any other example disclosed herein to achieve benefits as described in the present disclosure, including, for example, controlling depletion of electrolyte species.

Drawings

Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For brevity, reference numerals or features having previously described functions may be described in connection with other drawings in which they appear or may not.

FIG. 1 is a schematic partial cross-sectional view of an example of a nanopore sensor device disclosed herein;

FIG. 2A is an enlarged schematic view of a portion of a nanopore sensor device, wherein the cis trap includes a higher concentration of electrolyte species than the trans trap;

FIG. 2B is a schematic diagram of an example ion species drift and diffusion current of a nanopore sensor device, wherein the cis trap includes a higher concentration of electrolyte species than the trans trap;

FIG. 3 is an enlarged schematic view of a nanopore having a plurality of positively charged residues on its inner surface;

FIG. 4 is an enlarged schematic view of a portion of a nanopore sensor device in which an electrolyte solution includes a redox couple and redox inactive buffer ions having a diameter greater than the diameter of the constriction region of the nanopore;

FIGS. 5A and 5B are diagrams illustrating the inclusion of the same K in the cis-well and trans-well ⁺ Cl ^- A plot of simulated time dependence of current (5A; electrode current (pA) versus time (min)) and simulated material decomposition of current component (5B; current (pA) versus time (min)) for a comparative example nanopore sensor device of concentration;

FIGS. 6A and 6B are diagrams illustrating the inclusion of a higher K in the cis-well than in the trans-well ⁺ Cl ^- Analog time dependence of current of an exemplary nanopore sensor device of concentration (6A; electrode current(pA) vs. time (min)) and simulated mass decomposition of the current component (6B; current (pA) versus time (min));

FIGS. 7A and 7B are diagrams illustrating the inclusion of a higher K in the cis-well than in the trans-well ⁺ Cl ^- A plot of simulated time dependence of current (7A; electrode current (pA) versus time (min)) and simulated material decomposition of current components (7B; current (pA) versus time (min)) for another exemplary nanopore sensor device of concentration;

FIG. 8 is a graphical representation of the COSMOL analog domain used in example 4;

FIGS. 9A-9C show 3D finite element analysis results showing the calculated spatial distribution (logarithmic scale) of Cl: K ratios in the analog domain;

FIG. 10 is a graph showing the calculated dependence of Cl to K flux ratio through a nanopore on the magnitude of fixed charge density in the pore channel;

FIGS. 11A-11C are graphs showing calculated correlations of nanopore sensors with modified nanopores at various Cl to K flux ratios;

FIG. 12A is a graph showing current (nA, Y-axis) versus voltage (mV, X-axis) for a comparative MspA well;

FIG. 12B is a graph showing current (nA, Y-axis) versus voltage (mV, X-axis) for the MspA well of the first embodiment;

FIG. 13 is a graph showing average conductivity (S/m, Y axis) of the first example well and the comparative example well versus cis-trans voltage bias (mV, X axis) using KCl electrolytes of different concentrations;

FIG. 14A is a graph showing current (pA, Y axis) versus time (s, X axis) for a comparative MspA well; and is also provided with

Fig. 14B is a graph showing the current (pA, Y axis) versus time (s, X axis) of the MspA well of the first embodiment.

Detailed Description

Nanopore sequencing techniques use changes in electrical signals to distinguish nucleotide bases. The nanopore sensor device includes a cis well, a cis electrode, a plurality of trans wells, and a trans electrode associated with each of the plurality of trans wells. Each trans-well is separated from the cis-well by a lipid/polymer/solid-state membrane with nanopores. Thus, each trans-well is also associated with a respective nanopore. The faraday current between the cis and trans electrodes is established by the redox species with or without an electrolyte buffer.

In some examples, the electrode is active (i.e., the electrode actively participates in the redox reaction), and the reactive electrolyte species (i.e., redox anions) are consumed by plating out at the positive polarity counter electrode to support faraday current through the system. In some examples, the electrodes are passive (i.e., the electrodes do not actively participate in the redox reaction), and the reactive electrolyte species (i.e., redox pair ions) are consumed by oxidation at the positive polarity counter electrode to support faraday current through the system. In these examples, the redox couple is suspended in a redox inactive electrolyte buffer.

The counter electrode is located in a confined compartment having a finite volume, and therefore, in these cases, replenishment of the reactive electrolyte species in the trans trap depends on transport of the species from the cis trap through the nanopore. In existing nanopore sensor devices, the concentration of reactive electrolyte within the trans trap may become partially depleted, which reduces the operability of the nanopore sensor device.

In one example of an Ag/Cl redox system with an active electrode, the chloride anions may be partially depleted on the trans side due to plating onto the trans electrode and may not be completely replaced by transport of the chloride anions through the nanopore. For example, when two chloride ions are consumed by plating out at the counter electrode, one chloride ion enters the counter trap through the aperture. If the concentration of chloride ions in the trans trap is low, there is a reduced reactive electrolyte species in the trans trap. This reduces the ability to carry ionic current and thus leads to reduced signal levels and detection of the nanopore sensor device.

Because the reactive electrolyte species is replenished in a small fraction of the amount plated in existing nanopore sensor devices, depletion or partial depletion of the electrochemically active electrolyte species occurs in the trans trap over time. The partial consumption of the reactive electrolyte species depends on a number of factors, including the current through the nanopore and the size of the trans-trap (e.g., larger traps typically correlate to less reagent consumption, while smaller chambers typically correlate to more reagent consumption). The time for which the reactive electrolyte species in the trans-trap of existing nanopore sensor devices is fully depleted can be estimated by equation 1:

wherein V (cm) ³ ) Is the trans trap volume, C (mol/L) is the concentration of reactive electrolyte species in the trans trap at t=0, N _A Is the avogalileo number, q (coulomb or C) is the basic charge of the reactive electrolyte species, and i (a) is the nanopore current.

In existing nanopore sensor devices, partial consumption may be evidenced by a decrease in initial reagent concentration, where the decrease is greater than a factor of 10. In some cases, the reduction range is 20-fold to 100-fold. For example, the chlorine concentration of an electrolyte solution having an initial chlorine concentration of about 300mM in a 10 μm trans trap may be depleted to about 10mM, and thus the initial concentration reduced by a factor of about 30. For another example, the chlorine concentration of an electrolyte solution having an initial chlorine concentration of about 10mM in a 10 μm trans trap may be depleted to about 0.1mM, and thus the initial concentration is reduced by a factor of about 100. It will be appreciated that the partial consumption/depletion may be close to 100% (i.e. the partial consumption of electrolyte material remaining in the system is close to 0%), but will establish a balance between electrolyte anions and cations, even at such low levels of the partial consumption of material.

In the example of ferrocyanide/ferricyanide redox pairs in electrolyte buffers with passive electrodes, ferrocyanide ions (e.g., fe (CN) ₆ ^4- ) Oxidized to ferricyanide ions (e.g., fe (CN)) at the positive polarity counter electrode ₆ ^3- ). The reactive electrolyte species may be partially depleted on the trans side due to being depleted at the trans electrode, and may not beThe transport of the reactive electrolyte species through the nanopore is completely replaced.

Certain embodiments of the nanopore sensor devices and methods disclosed herein reduce depletion of reactive electrolyte species at the trans-well of the nanopore sensor device. In these examples, a decrease in the initial reactive electrolyte species concentration at the trans trap may still be present; however, this reduction is less than 10% and possibly less than 1%. Thus, there is more reactive electrolyte species over time (e.g., as compared to existing nanopore sensor devices provided above), and thus depletion of the reactive electrolyte species is reduced. In certain examples, depletion of the reactive electrolyte species is reduced by inhibiting transmission of redox cations, buffer anions, or both through the nanopores of the nanopore sensor device. In these particular examples, a reduction in the transport of redox cations is achieved using an unbalanced electrolyte concentration, modified nanopores, and/or charges induced in the nanopores. In these particular examples, a reduction in anion transport is achieved using an unbalanced electrolyte concentration and/or utilizing a large volume of buffer anions. Reducing the transport of redox cations and/or buffer anions through the nanopore results in a greater amount of ionic charge being carried by the reactive electrolyte species. Thus, an increased amount of reactive electrolyte species is transported from the cis-well to the trans-well. Thus, the depletion of reactive electrolyte species from the transwells of the nanopore sensor device is reduced and the lifetime of the nanopore sensor device is prolonged.

As described above, nanopore sequencing techniques use changes in electrical signals to distinguish nucleotide bases. Depletion of the reactive electrolyte species in the trans trap reduces the ion current and reduces the signal detected by the nanopore sensor device. By increasing the replenishment of the reactive electrolyte species from the cis-well to the trans-well, the lifetime of the nanopore sensor device can be increased.

Definition of the definition

It will be understood that the terms used herein, unless otherwise indicated, are to be understood to have their ordinary meaning in the relevant art. Several terms used herein and their meanings are listed below.

The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The terms including, comprising, housing and the various forms of these terms are synonymous with each other and are intended to be equally broad.

The terms top, bottom, lower, upper, etc. are used herein to describe the nanopore sensor device and/or various components of the nanopore sensor device. It should be understood that these directional terms are not intended to imply a particular orientation, but rather are used to designate relative orientations between components. The use of directional terms should not be construed to limit the examples disclosed herein to any particular orientation.

The terms first, second, etc. are also not intended to imply a particular orientation or order but are used to distinguish one element from another.

It is to be understood that the ranges provided herein include the specified ranges and any value or subrange within the specified ranges, as if such value or subrange were explicitly recited. For example, a range of about 50mM to about 500mM should be construed to include not only the explicitly recited limits of about 50mM to about 500mM, but also to include individual values (such as about 100mM, about 335mM, about 400.5mM, about 490mM, etc.) and subranges (such as about 75mM to about 475mM, about 200mM to about 300mM, etc.). Furthermore, when values are described using "about" and/or "substantially," they are intended to cover minor variations (up to +/-10%) from the stated values.

As used herein, the terms "fluidly connected," "fluidly coupled," and the like refer to two spatial regions that are connected together such that a liquid or gas can flow between the two spatial regions. For example, the cis-trap may be fluidly connected to the trans-trap or traps such that at least a portion of the electrolyte solution may be transferred between the connected traps. The two spatial regions may be in fluid communication through a nanopore or through one or more valves, restrictors, or other fluidic components for controlling or regulating ion transfer through the system.

As used herein, the term "interstitial regions" refers to regions in a substrate/solid support or film, or regions on a surface that separate other regions, areas, features associated with the support or film or surface. For example, a gap region of the membrane may separate one nanopore of the array from another nanopore of the array. For another example, a gap region of the substrate may separate one transwell from another transwell. The two regions that are separated from each other may be discrete, i.e., lack physical contact with each other. In many examples, the interstitial regions are continuous, while the regions are discrete, e.g., for a plurality of nanopores defined in an otherwise continuous membrane, or for a plurality of wells defined in an otherwise continuous support. The separation provided by the gap region may be a partial separation or a complete separation. The interstitial regions may have a surface material that is different from a surface material of the features defined in the surface. For example, the surface material at the interstitial regions may be a lipid material, and the nanopores formed in the lipid material may have an amount or concentration of polypeptide that exceeds the amount or concentration present at the interstitial regions. In some examples, the polypeptide may not be present at the gap region.

As used herein, the term "membrane" refers to an impermeable or semi-permeable barrier or other sheet separating two liquid/gel chambers (e.g., cis-well and trans-well) that can house the same composition or different compositions therein. The permeability of a membrane to any given substance depends on the nature of the membrane. In some examples, the membrane may be ion, current, and/or fluid impermeable. For example, the lipid membrane may be ion impermeable (i.e., not allow any ion transport therethrough), but may be at least partially permeable to water (e.g., a water diffusivity in the range of about 40 μm/s to about 100 μm/s). For another example, a synthetic/solid film (such as silicon nitride) may be ion, charge, and fluid impermeable (i.e., diffusion of all of these substances is zero). Any membrane may be used in accordance with the present disclosure so long as the membrane is capable of including a transmembrane nanoscale opening (e.g., a nanopore) and is capable of maintaining a potential difference across the membrane. The film may be a single layer or a multilayer film. The multilayer film includes two or more layers, each of which is an impermeable or semi-permeable material.

The membrane may be formed from a material of biological or non-biological origin. A material of biological origin refers to a material derived from or isolated from a biological environment (such as an organism or cell), or a synthetically manufactured form of a biologically useful structure (e.g., a biomimetic material).

Exemplary membranes made from materials of biological origin include monolayers formed from bolapid (bolapid). Another exemplary membrane made from a material of biological origin includes a lipid bilayer. Suitable lipid bilayers include, for example, membranes of cells, membranes of organelles, liposomes, planar lipid bilayers, and supported lipid bilayers. The lipid bilayer may for example be formed of two opposing phospholipid layers arranged such that their hydrophobic tail groups face each other to form a hydrophobic interior, while the hydrophilic head groups of the lipid face outwards towards the aqueous environment on each side of the bilayer. Lipid bilayers can also be formed, for example, by a process in which a lipid monolayer is carried on an aqueous solution/air interface through either side of a pore perpendicular to the interface. Lipids are typically added to the surface of an aqueous electrolyte solution by first dissolving the lipid in an organic solvent and then evaporating a drop of solvent on the surface of the aqueous solution on either side of the pore. Once the organic solvent at least partially evaporates, the solution/air interface on either side of the pore physically moves up and down through the pore until a bilayer is formed. Other suitable methods of bilayer formation include tip dipping, patch clamp coating bilayers and liposomal bilayers. Any other method for obtaining or generating lipid bilayers may also be used.

Materials of non-biological origin may also be used as membranes. Some of these materials are solid materials and may form solid films, and others of these materials may form thin liquid films or membranes. The solid film may be a single layer, such as a coating or film on a support substrate (i.e., a solid support), or may be a freestanding element. The solid state film may also be a composite of multiple layers of material in a sandwich configuration. Any non-biological source of material may be used as long as the resulting membrane is capable of including transmembrane nanoscale openings and is capable of retaining a transmembrane membraneIs only needed. The film may comprise an organic material, an inorganic material, or both. Examples of suitable solid state materials include, for example, microelectronic materials, insulating materials (e.g., silicon nitride (Si ₃ N ₄ ) Alumina (Al) ₂ O ₃ ) Hafnium oxide (HfO) ₂ ) Tantalum pentoxide (Ta) ₂ O ₅ ) Silicon oxide (SiO) ₂ ) Etc.), some organic and inorganic polymers (e.g., polyamides, plastics such as Polytetrafluoroethylene (PTFE), or elastomers such as two-component addition cure silicone rubber), and glass. Further, the solid-state film may be made of a single layer of graphene (which is carbon atoms densely packed into atomic-scale sheets of a two-dimensional honeycomb lattice), multiple layers of graphene, or one or more layers of graphene mixed with one or more layers of other solid-state materials. The graphene-containing solid state membrane may include at least one graphene layer that is a graphene nanoribbon or a graphene nanogap, which may be used as an electrical sensor to characterize a target polynucleotide. The solid state film may be prepared by any suitable method. For example, graphene films may be fabricated by Chemical Vapor Deposition (CVD) or exfoliation from graphite. Examples of suitable thin liquid film materials that may be used include diblock copolymers, triblock copolymers, such as amphiphilic PMOxa-PDMS-PMOxa ABA triblock copolymers.

As used herein, the term "nanopore" is intended to mean a hollow structure that is discrete from a membrane and extends through the membrane, allowing ions, current, and/or fluid to pass from one side of the membrane to the other side of the membrane. For example, a membrane that inhibits the passage of ions or water-soluble molecules may include a nanopore structure that extends through the membrane to allow the passage of ions or water-soluble molecules from one side of the membrane (through nanoscale openings/channels that extend through the nanopore structure) to the other side of the membrane. The diameter of the nanoscale openings/channels may vary along their length (i.e., from one side of the membrane to the other side of the membrane), but at any point are on the nanoscale (i.e., about 1nm to about 100nm, or to less than 1000 nm). Examples of nanopores include, for example, biological nanopores, solid state nanopores, and biological and solid state hybrid nanopores.

As used herein, the term "diameter" is intended to mean the longest straight line inscribable in the cross-section of the nanoscale opening through the centroid of the cross-section of the nanoscale opening. It should be appreciated that the nanoscale openings may or may not have a circular or substantially circular cross-section (the cross-section of the nanoscale openings being substantially parallel to the cis/trans electrodes). Furthermore, the cross-section may be regular or irregularly shaped.

As used herein, the term "biological nanopore" is intended to mean a nanopore whose structural portion is made of a material of biological origin. Biological origin refers to materials derived from or isolated from a biological environment (such as an organism or cell), or synthetically manufactured forms of bioavailable structures. Biological nanopores include, for example, polypeptide nanopores and polynucleotide nanopores.

As used herein, the term "polypeptide nanopore" is intended to mean a protein/polypeptide that extends through a membrane and allows ions and/or fluids to flow from one side of the membrane to the other side of the membrane. The polypeptide nanopore may be a monomer, a homopolymer or a heteropolymer. The structure of polypeptide nanopores includes, for example, alpha-helical bundle nanopores and beta-barrel nanopores. Exemplary polypeptide nanopores include alpha-hemolysin, mycobacterium smegmatis (Mycobacterium smegmatis) porin a (MspA), gramicidin a, maltose porin, ompF, ompC, phoE, tsx, F-pili, and the like. The protein α -hemolysin naturally occurs in the cell membrane where it serves as a pathway for ion or molecule transport into and out of the cell. Mycobacterium smegmatis porin A (MspA) is a porin produced by Mycobacteria that allows hydrophilic molecules to enter bacteria. MspA forms tightly interconnected octamers and transmembrane β -buckets, which resemble goblets and house a central channel/bore.

Polypeptide nanopores may be synthetic. The synthetic polypeptide nanopore includes a proteinaceous amino acid sequence that does not exist in nature. The proteinaceous amino acid sequence may include some of the amino acids known to be present but not forming the basis of the protein (i.e., non-proteinogenic amino acids). The proteinaceous amino acid sequence may be synthesized artificially rather than expressed in an organism, and then purified/isolated.

As used herein, the term "polynucleotide nanopore" is intended to include polynucleotides that extend through a membrane and allow ions and/or fluids to flow from one side of the membrane to the other side of the membrane. The polynucleotide pore may include, for example, a polynucleotide fold (e.g., nanoscale folding of DNA to create a nanopore).

Also as used herein, the term "solid state nanopore" is intended to mean a nanopore whose structural portion includes a material of non-biological origin (i.e., not of biological origin). The solid state nanopores may be formed from inorganic or organic materials. Solid state nanopores include, for example, silicon nitride nanopores, silicon dioxide (SiO) ₂ ) Nanopores and graphene nanopores.

The nanopores disclosed herein may be hybrid nanopores. "hybrid nanopore" refers to a nanopore comprising materials of both biological and non-biological origin. Examples of hybrid nanopores include polypeptide-solid state hybrid nanopores and polynucleotide-solid state nanopores.

As used herein, the term "nanopore sensor device" or "nanopore sequencer" refers to any device disclosed herein that can be used for nanopore sequencing. In examples disclosed herein, during nanopore sequencing, a nanopore is immersed in an example of an electrolyte disclosed herein, and a potential difference is applied across the membrane. In an example, the potential difference is an electrical potential difference or an electrochemical potential difference. A potential difference may be applied across the membrane by a voltage source that injects or applies a current to at least one ion of the electrolyte contained in the cis-well or the one or more trans-wells. The electrochemical potential difference can be established by a combination of the difference in ion composition of the cis and trans traps and the potential. The different ion compositions may be, for example, different ions in each trap or different concentrations of the same ions in each trap.

Applying a potential difference across the nanopore may force the nucleic acid to translocate through the nanopore. One or more signals corresponding to translocation of the nucleotide through the nanopore are generated. Thus, when a target polynucleotide or mononucleotide or a probe derived from a target polynucleotide or mononucleotide passes through a nanopore, the current across the membrane changes due to, for example, base-dependent (or probe-dependent) blocking of the constriction. The signal from this current change may be measured using any of a variety of methods. Each signal is unique to the type of nucleotide (or probe) in the nanopore, such that the resulting signal can be used to determine a characteristic of the polynucleotide. For example, the identity of one or more nucleotide (or probe) species that produce the characteristic signal may be determined.

As used herein, "nucleotide" includes nitrogen-containing heterocyclic bases, sugars, and one or more phosphate groups. Nucleotides are monomeric units of a nucleic acid sequence. Examples of the nucleotide include, for example, ribonucleotide or deoxyribonucleotide. In Ribonucleotides (RNA), the sugar is ribose, and in Deoxyribonucleotides (DNA), the sugar is deoxyribose, i.e. a sugar in ribose that lacks a hydroxyl group present at the 2' position. The nitrogen-containing heterocyclic base may be a purine base or a pyrimidine base. Purine bases include adenine (a) and guanine (G) and modified derivatives or analogues thereof. Pyrimidine bases include cytosine (C), thymine (T) and uracil (U) and modified derivatives or analogues thereof. The C-1 atom of deoxyribose is bonded to N-1 of pyrimidine or N-9 of purine. The phosphate groups may be in the form of mono-, di-or tri-phosphate. These nucleotides are natural nucleotides, but it is further understood that non-natural nucleotides, modified nucleotides or analogs of the foregoing may also be used.

As used herein, the term "signal" is intended to mean an indicator representing information. Signals include, for example, electrical signals and optical signals. The term "electrical signal" refers to an indicator representing the electrical quality of information. The indicator may be, for example, a current, voltage, tunneling, resistance, potential, conductance, capacitance, frequency, or other change in electrical waveform.

The term "substrate" refers to a rigid solid support that is insoluble in aqueous liquids and that is incapable of passing liquids without pores, ports, or other similar liquid conduits. In examples disclosed herein, a substrate may have a well or chamber defined therein. Examples of suitable substrates include glass and modified or functionalized glass, plastics (including acrylics, polystyrene, and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (PTFE) (such as those from Chemours)) Cycloolefin/cycloolefin Polymer (COP) (such as +.>) Polyimide, etc.), nylon, ceramic, silica or silica-based materials, silicon and modified silicon, carbon, metal, inorganic glass, and fiber bundles.

"stimulus" refers to an electronic device that provides a stimulus that causes ionic current to flow through a nanopore. In one example, the stimulus source may be a current source or a voltage source coupled to the cis and/or trans electrodes. In another example, the stimulus may be any source that generates an electric field between the cis-well and the trans-well.

As used herein, the terms "well," "cavity," and "chamber" are used synonymously and refer to discrete features defined in a device that can contain a fluid (e.g., liquid, gel, gas). A "cis-well" is a common chamber containing or defined in part by a cis-electrode and is also fluidly connected to each of a plurality of trans-wells by a respective nanopore. Examples of arrays of the inventive apparatus may have one cis well or multiple cis wells. Each transwell is a single chamber that includes or is partially defined by its own transelectrode and is also in fluid connection with one of the cis wells. Each of the retrograde wells is electrically isolated from each of the other retrograde wells. In some examples, each transwell is connected to a respective stimulus source and to a respective amplifier (e.g., an Axopatch 200B amplifier) to amplify an electrical signal passing through a respective nanopore associated with each transwell. In other examples, the transwells are connected to a single stimulus that individually addresses the transwells via multiplexing. Furthermore, it should be appreciated that the cross-section of the well taken parallel to the surface of the substrate at least partially defining the well may be curved, square, polygonal, hyperbolic, conical, angular, etc.

In view of the above definitions, the aspects and examples set forth herein and recited in the claims can be understood.

Nanopore sensor device

Referring now to fig. 1, an example of a nanopore sensor device 10 is shown. The nanopore sensor device 10 includes: one or more cis traps 12; a cis electrode 14; a plurality of trans traps 16, each trans trap of the plurality of trans traps 16 being separated from one or more cis traps 12 by a lipid/polymer/solid film 18 having a nanopore 20; a plurality of counter electrodes 22, each counter electrode of the plurality of counter electrodes 22 being associated with one of the plurality of counter wells 16. The exemplary nanopore sensor device 10 also includes an electrolyte solution 24 in the cis well 12 and the trans well 16. Different examples of the device 10 include different examples of the electrolyte solution 24, which will be described with reference to fig. 2A through 4.

As shown in fig. 1, the nanopore sensor device 10 includes a substrate 26. The substrate 26 may include a plurality of trans wells 16 defined therein. Each of the trans traps 16 can be fluidly connected to the common cis trap 12 through a respective nanopore 20. Although one common cis-well 12 is shown in fig. 1, it should be understood that the nanopore sensor device 10 may include several cis-wells 12 that are fluidly isolated from each other and fluidly connected to a corresponding set of trans-wells 16 defined in the substrate 26. Multiple cis-traps 12 may be required, for example, to be able to measure multiple samples on a single substrate 26.

Fluid communication through the nanopore 20 is represented by arrows in fig. 1. Additionally, as shown in fig. 1, a membrane 18 may be positioned on the substrate 26 between the cis well 12 and the trans well 16, and a nanopore 20 may be positioned in and extend through the membrane 18 to establish a fluid connection between the cis well 12 and the trans well 16.

The cis trap 12 is a fluid chamber defined on a portion of the substrate 26 by a sidewall 28 connected to the substrate 26. In some examples, the side walls 28 and the base 26 may be integrally formed such that they 28, 26 are formed from a continuous sheet of material (e.g., glass or plastic). In other examples, the sidewall 28 and the base 26 may be separate components coupled to one another. In an example, the sidewalls 28 are a photo-patternable polymer.

In the example shown in fig. 1, the cis well 12 has an inner wall 30 defined by the side wall 28, an upper surface 32 defined by the cis electrode 14, and a lower surface 34 defined by the membrane 18. Thus, the cis well 12 is formed within the space defined by the cis electrode 14, the portion of the substrate 26, and the membrane 18. It should be appreciated that the lower surface 34 has an opening through the nanopore 20 positioned in the membrane 18. The cis-well 12 may have any suitable dimensions. In an example, the cis well 12 may be in the range of about 1mm by 1mm to about 5mm by 5 mm.

The cis electrode 14 (whose interior surface is the upper surface 32 of the cis well 12) may be physically connected to the sidewall 28. The cis electrode 14 may be physically attached to the sidewall 28, such as by an adhesive or another suitable fastening mechanism. The interface between the cis electrode 14 and the sidewall 28 may seal the upper portion of the cis well 12.

The cis-electrode 14 used depends at least in part on the electrolyte species in the electrolyte solution 24 (or 24A, 24B, 24C as described herein). In some examples, the cis electrode 14 may be an active electrode that participates in a chemical reaction with an electrochemically active electrolyte species, and may be oxidized or reduced in a half-cell reaction. Examples of the active electrode include silver (Ag), copper (Cu), zinc (Zn), lead (Pb), and the like. In other examples, the cis electrode 14 may be an inactive (or inert) electrode that transfers electrons rather than exchanging ions with the electrolyte solution 24. Examples of the inactive electrode include platinum (Pt), carbon (C) (e.g., graphite, diamond, etc.), gold (Au), rhodium (Rh), etc. In the use of electroactive anions (e.g., chloride, cl ^- ) In the example of the nanopore sensor 10 of electrolyte solution 24, the cis electrode 14 may be a silver/silver chloride (Ag/AgCl) electrode.

The cis-well 12 is capable of maintaining the electrolyte solution 24 in contact with the nanopore 20. In an example, the cis well 12 is in contact with the array of nanopores 20 and is therefore able to hold the modified electrode 24 in contact with each of the nanopores 20 in the array.

As shown in fig. 1, the nanopore sensor device 10 includes a plurality of transwells 16. Each transwell 16 is a fluid chamber defined in a portion of the substrate 26. In general, the trans trap 16 may extend through the thickness of the substrate 26 and may have openings at opposite ends (e.g., top and bottom ends 36, 38) of the substrate 26. In the example shown in fig. 1, each of the anti-wells 16 has sidewalls 40 defined by the substrate 26 and/or a gap region 42 of the substrate 26, a lower surface 44 defined by the anti-electrode 22, and an upper surface 46 defined by the film 18. Thus, each of the anti-wells 16 is formed within the space defined by the anti-electrode 22, another portion of the substrate 26 and/or the gap region 42, and the film 18. It should be appreciated that the upper surface 46 has an opening through the nanopore 20 positioned in the membrane 18.

The counter electrode 22 (whose inner surface is the lower surface 44 of the counter well 16) may be physically connected to the substrate 26 (e.g., to the gap region 42 or to the inner wall of the substrate 26). The counter electrode 22 may be fabricated during formation of the substrate 26 (e.g., during formation of the counter well 16). Micro-fabrication techniques that may be used to form the substrate 26 and the counter electrode 22 include photolithography, metal deposition and lift-off, dry and/or spin-on film deposition, etching, and the like. The interface between the counter electrode 22 and the substrate 26 may seal a lower portion of the counter well 16.

The counter electrode 22 used depends at least in part on the electrolyte species in the electrolyte solution 24 (or 24A, 24B, 24C as described herein). The counter electrode 22 may be an active electrode that participates in a chemical reaction with an electrochemically active electrolyte substance and may be oxidized or reduced in a half-cell reaction. Any of the examples of active electrodes described herein for cis electrode 14 may be used as trans electrode 22. In other examples, the counter electrode 22 may be an inactive (or inert) electrode that transfers electrons rather than exchanging ions with the electrolyte solution 24. Any of the examples set forth herein for cis electrode 14 may be used as trans electrode 22. In the use of electroactive anions (e.g., chloride, cl ^- ) In the example of the nanopore sensor 10 of electrolyte solution 24, the counter electrode 22 may be a silver/silver chloride (Ag/AgCl) electrode.

Many different layouts of the trans-well 16 are contemplated, including regular, repeating, and irregular patterns. In the example, the trans-traps 16 are arranged in a hexagonal grid to achieve a close packing and improved density. Other layouts may include, for example, a straight line (i.e., rectangular) layout, a triangle layout, and so forth. As an example, the layout or pattern may be in the x-y format of the transwells 16 in rows and columns. In some other examples, the layout or pattern may be a repeating arrangement of the retrograde well 16 and/or the gap region 42. In still other examples, the layout or pattern may be a random arrangement of the retrograde well 16 and/or the gap region 42. The pattern may include dots, posts, bars, swirls, lines, triangles, rectangles, circles, arcs, corrugations, grids, diagonals, arrows, squares, and/or cross-hatching.

The layout may be characterized relative to the density of the anti-wells 16 (i.e., the number of anti-wells 16 in a defined area of the substrate 26). For example, the trans-well 16 may be between about 10 wells per mm ² Up to about 1,000,000 wells per mm ² Densities within the range exist. The density may be adjusted to different densities including, for example, at least about 10 per mm ² About 5,000 per mm ² About 10,000 per mm ² About 10 ten thousand per mm ² Or greater density. Alternatively or in addition, the density may be adjusted to not more than about 1,000,000 wells per mm ² About 10 ten thousand wells per mm ² About 10,000 wells per mm ² About 5,000 wells per mm ² Or less. It should also be appreciated that the density of the trans-well 16 in the support 26 may be between one value selected from the lower and upper values of the ranges described above.

The layout may also or alternatively be characterized in terms of average pitch, i.e., the spacing from the center of a nanopore 20 to the center of an adjacent nanopore 20 (center-to-center spacing). The pattern may be regular such that the coefficient of variation around the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In an example, the average pitch may be in the range of about 100nm to about 500 μm. The average pitch may be, for example, at least about 100nm, about 5 μm, about 10 μm, about 100 μm, or more. Alternatively or in addition, the average pitch may be, for example, up to about 500 μm, about 100 μm, about 50 μm, about 10 μm, about 5 μm, or less. The average pitch of an exemplary array of nanopores 20 including a particular pattern may be between one value selected from the lower and upper values of the ranges described above. In an example, the array has an average pitch (center-to-center spacing) of about 10 μm.

The trans-well 16 may be a micro-well (having at least one dimension on the micrometer scale, e.g., about 1 μm to 1000 μm, but excluding 1000 μm) or a nano-well (having at least one dimension on the nanometer scale, e.g., about 10nm to 1000nm, but excluding 1000 nm). Each trans-well 16 may be characterized by its aspect ratio (e.g., width or diameter divided by depth or height, respectively).

In an example, the aspect ratio of each trans well 16 may be in the range of about 1:1 to about 1:5. In another example, the aspect ratio of each trans well 16 may be in the range of about 1:10 to about 1:50. In an example, the aspect ratio of the trans well 16 is about 3.3.

The depth/height and width/diameter may be selected to achieve a desired aspect ratio. The depth/height of each trans well 16 may be at least about 0.1 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or in addition, the depth may be up to about 1,000 μm, about 100 μm, about 10 μm, about 1 μm, about 0.1 μm, or less. The width/diameter of each trans well 16 may be at least about 50nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 10 μm, about 100 μm, or more. Alternatively or in addition, the width/diameter may be up to about 1,000 μm, about 100 μm, about 10 μm, about 1 μm, about 0.5 μm, about 0.1 μm, about 50nm, or less.

Each trans well 16 has an opening (e.g., an opening facing cis well 12) that is large enough to accommodate at least a portion of membrane 18 and its associated nanopore 20. For example, the ends of the nanopores 20 may extend through the membrane 18 and into the openings of the trans-well 16.

Various techniques may be used to fabricate cis-well 12 and trans-well 16, including, for example, photolithography, nanoimprint lithography, stamping techniques, imprint techniques, molding techniques, microetching techniques, and the like. As will be appreciated by those skilled in the art, the technique used will depend on the composition and shape of the substrate 26 and the sidewalls 30. In an example, the cis well 12 may be defined by a sidewall 30 at the end 26 of the substrate 26, and the trans well 16 may be defined by the substrate 26.

The membrane 18 may be any of the impermeable or semi-permeable materials described herein. The membrane 18 is positioned between the cis 12 and trans 16 traps and thus provides a barrier between the traps 12, 16. The film may be positioned on the gap region 42 of the substrate 26.

The nanopore 20 may be any of the biological nanopores, solid state nanopores, and hybrid nanopores described herein. In one example, the nanopore 20 is a modified nanopore 20' as described with reference to fig. 3. As described herein, each nanopore 20 fluidly connects a respective one of the trans traps 16 with the cis trap 12. Thus, the ratio of nanopore 20 to trans well 16 is 1:1.

The nanopore 20 has two open ends and a hollow core or pore connecting the two open ends. The wall of the hollow core or bore is the inner surface 48 of the nanopore 20. When inserted into the membrane 18, one of the open ends of the nanopore 20 faces the cis well 12 and the other of the open ends of the nanopore 20 faces the trans well 16 and is aligned with at least a portion of the opening of the trans well 16. The hollow core of the nanopore 20 enables fluid connection between the wells 12, 16. The diameter of the hollow core may be in the range of about 1nm to 1 μm and may vary along the length of the nanopore 20. In some examples, the open end facing cis-well 12 may be larger than the open end facing trans-well 16. In other examples, the open end facing cis-well 12 may be smaller than the open end facing trans-well 16.

The nanopore 20 may be inserted in the membrane 18, or the membrane 18 may be formed around the nanopore 20. In an example, the nanopore 18 may insert itself into a formed lipid bilayer (one example of a membrane 18). For example, the nanopore 20 in monomeric or polymeric form (e.g., octamer) may insert itself into a lipid bilayer and assemble into a transmembrane pore. In another example, the nanopore 20 may be added to the ground side of the lipid bilayer at a desired concentration, where the nanopore inserts itself into the lipid bilayer. In yet another example, a lipid bilayer may be formed across pores in a Polytetrafluoroethylene (PTFE) film and positioned between a cis-well and a trans-well. The nanopore 20 may be added to the grounded cis compartment and may itself be inserted into the lipid bilayer at the region where the PTFE pore is formed. In yet another example, the nanopores 20 may be tethered to a solid support (e.g., silicon oxide, quartz, indium tin oxide, gold, polymer, etc.). The tethering molecule (which may be part of the nanopore 20 itself or may be attached to the nanopore 20) may attach the nanopore 20 to a solid support. Attachment via tethering molecules may allow a single aperture 20 to be fixed (e.g., between two chambers/wells). A lipid bilayer may then be formed around the nanopore 20.

The nanopore sensor device 10 includes an electrolyte solution 24 in a cis-well 12 and a trans-well 16. Different examples of the device 10 disclosed herein include different examples of the electrolyte solution 24, which are described in more detail herein with reference to fig. 2A-4.

The nanopore sensor device 10 also includes electronics that individually and/or collectively address each of the counter electrodes 22. As described herein, each of the transwell electrodes 22 is associated with a respective transwell 16 and a respective nanopore 20. Some of the electronics are schematically shown in circuit diagram form in fig. 1. These electronics include at least a stimulus source 52 and a controller 50. The stimulus 52 is coupled to each of the plurality of counter electrodes 22 individually (as shown in fig. 1) or via multiplexing, and the stimulus 52 will cause current to flow through one or more of the nanopores 20 by addressing the counter electrode 22 associated with the respective nanopore 20. The controller 50 is coupled to the stimulus source 52, and the controller 50 is configured to individually/selectively address one of the plurality of counter electrodes 22 (using the stimulus source) to flow an ionic current through the nanopore 20 connected to the addressed counter electrode 22. In one example, each of the counter electrodes 22 is electrically connected to its own set of electronics, including a stimulus source 52 and a controller 50. In another example, each of the counter electrodes 22 is electrically connected to a single stimulus source 52 and controller 50, which are connected to a multiplexer (not shown). As shown in fig. 1, the electronic device may further include an amplifier 54 that amplifies the electrical signal passing through the corresponding nanopore 20 associated with the addressed counter electrode 22.

Electrolyte concentration

Referring now to fig. 2A, one example of a nanopore sensor device 10 includes an electrolyte solution 24A in cis-well 12 and an electrolyte solution 24B in each trans-well in trans-well 16, where the electrolyte solutions 24A, 24B have different concentrations of reactive electrolyte species therein (made up of C ⁺ 、A ^- Representation). For example, C ⁺ Can represent redox cations or redox agent ions, and A ^- May represent a redox anion or a redox oxidant ion. More specifically, electrolyte C in electrolyte solution 24A within cis-well 12 ⁺ 、A ^- Is higher than the electrolyte C in the electrolyte solution 24B in the trans trap 16 ⁺ 、A ^- Is a concentration of (3). In these examples, the electrodes 14, 22 may be active electrodes or passive electrodes.

Although the reactive electrolyte species C in the electrolyte solutions 24A, 24B ⁺ 、A ^- The types of the electrolyte solutions 24A, 24B are the same, but the reactive electrolyte species C ⁺ 、A ^- Is different in concentration. More specifically, the reactive electrolyte species C to be introduced or contained in the electrolyte solution 24A in the cis trap 12 ⁺ 、A ^- Is higher than the concentration of the reactive electrolyte species C in the electrolyte solution 24B to be introduced or contained in the trans trap 16 ⁺ 、A ^- Is a concentration of (3). In some examples, electrolyte C in electrolyte solution 24A (for cis-well 12) ⁺ 、A ^- Concentration and electrolyte C in electrolyte solution 24B (for trans trap 16) ⁺ 、A ^- The ratio of concentrations is in the range of about 10:1 to about 3:1. In one example, electrolyte C in electrolyte solution 24A (for cis-well 12) ⁺ 、A ^- Concentration and electrolyte C in electrolyte solution 24B (for trans trap 16) ⁺ 、A ^- The ratio of concentrations is in the range of about 8:1 to about 5:1. In some examples, reactive electrolyte species C in electrolyte solution 24A ⁺ 、A ^- The concentration may range from about 300mM to about 1000mM, and electrolyte C in electrolyte solution 24B ⁺ 、A ^- Concentration can beAnd thus about 100mM.

Each of the electrolyte solutions 24A, 24B further includes a polar solvent. In one example, the polar solvent is water. Electrolyte C of different concentrations ⁺ 、A ^- Dissolved in a polar solvent to form the corresponding electrolyte solutions 24A, 24B.

In some examples, the nanopore sensor device 10 may store an electrolyte solution 24B having the same concentration of electrolyte C in the cis 12 and trans 16 wells ⁺ 、A ^- . When it is desired to utilize the nanopore sensor device 10, for example, in a sensing operation, additional amounts of reactive electrolyte species may be introduced or exchanged into the electrolyte solution 24A to form a higher concentration of reactive electrolyte species C in the cis-well 12 ⁺ 、A ^- (e.g., via fluid inlet 56).

Thus, one example disclosed herein is a nanopore sensor package that includes an additional amount of reactive electrolyte species C to be introduced into one or more cis wells 12 through fluid inlet 56 ⁺ A-such that upon initial cycling of the nanopore sensor device 10, one or more of the cis traps 12 contains a higher concentration of electrolyte C ⁺ 、A ^- And the plurality of trans traps 16 contain a lower concentration of electrolyte C ⁺ 、A-。

When the respective electrolyte solutions 24A, 24B are contained within the cis-well 12 and trans-well 16, nucleotide samples may be added to the cis-well 12, for example, via fluid inlet/outlet ports 56 and 58. The controller 50 may then be used to activate the stimulus source 52 to individually/selectively address one of the plurality of counter electrodes 22. A stimulus source 52 (e.g., a current source, a voltage source) causes an ionic current to flow through the nanopore 20 connected to the addressed counter electrode 22. Due to the highly reactive electrolyte species C in the cis-trap 12 ⁺ 、A ^- Concentration, ionic current includes reactive electrolyte species C ⁺ ，A ^- A quantity a translocated to the addressed transwell 16 through the nanopore 20 ^- (e.g., redox anions, redox oxidant ions), the A ^- Is of the amount ofIn reactive electrolyte substance C ⁺ 、A ^- C translocated from addressed trans-well 16 through nanopore 20 ⁺ (e.g., redox cations, redox reagent ions).

In one example, stimulus 52 applies a voltage bias (using electrodes 14, 22) between cis-well 12 and at least one of the plurality of trans-wells 16 and thus across membrane 18. The applied voltage bias may be positive polarity of the counter electrode 22 to attract negatively charged compounds (such as negatively charged nucleotides, negatively charged tags/labels) in the cis-well 12 toward the nanopore 20, and/or negative polarity of the counter electrode 22 to repel negatively charged compounds (such as negatively charged nucleotides, negatively charged tags/labels) in the cis-well 12 away from the nanopore 20. In an example, the voltage bias between the cis well 12 and the addressed one of the plurality of trans wells 16 is in the range of about-1V to about 1V. Any voltage bias within a given range may be applied when the electrolyte solutions 24A, 24B are present in the respective wells 12, 16. In some examples, the nanopore sensor device 10 operates in a bipolar mode (e.g., alternating current) providing a negative bias and a positive bias to the counter electrode 22. In some examples, the nanopore sensor device 10 operates in a monopolar mode (e.g., direct current), thereby providing a negative bias to the counter electrode 22.

The cis-trans voltage bias causes ionic current to flow through the nanopore 20 associated with the addressed trans electrode 22. In one example, the current passing through the nanopore 20 forces the corresponding nucleotide and/or tag/label to react with the charge-carrying reactive electrolyte species a ^- Together translocate into the nanopore 20. When a nucleotide and/or label passes through the nanopore 20, the current through the barrier changes, for example, due to blockage of the nanopore constriction 60 (see fig. 3), a change in resistance of the nanopore 20, and a change in capacitance of the nanopore 20. The signal from this change may be measured using an amplifier 54 or another known signal detection device. Depending on the bias, charge carrying reactive electrolyte species A ^- May be transferred from cis well 14 to trans well 16.

In this example, voltage polarity is typically applied such that negatively charged nucleic acids and/or labels/tags are electrophoretically driven into nanopore 20. In some cases, the voltage may be reduced or the polarity reversed to facilitate proper function.

When the reactive electrolyte material C ⁺ 、A ^- The concentration of reactive electrolyte species C is higher in cis-well 12 than in trans-well 16 ⁺ The drift and diffusion currents are in opposite directions. A schematic diagram of this situation is shown in fig. 2B. Electrolyte substance C having higher reactivity on cis side 12 upon initiation of sensing ⁺ At a concentration, an initial reactive electrolyte species C at t=0 ⁺ The current is reduced and even eliminated. In this way, starting from t=0, the reactive electrolyte species a ^- Is forced to carry most of the ion current, thus alleviating the initial drop in current and the total current will remain more stable throughout the sensing run. The lifetime of the nanopore sensor device 10 may be extended, in part, because a greater proportion of the current is forced through the nanopore 20 to be additionally bound by the reactive electrolyte species a ^- Is carried by the patient. In addition, the reactive electrolyte species A added on the cis side 12 ^- Concentration for increasing reactive electrolyte species A ^- The current is diffused so that a higher total current is maintained.

Modified nanopores

Another example of a nanopore sensor device 10 includes modified nanopores in the location of each of the nanopores 20. An example of a modified nanopore 20' is shown in fig. 3.

Modified nanopore 20' is a protein or solid state nanopore whose inner surface 48 has been modified to introduce at least one fixed positive charge (referred to as a positively charged residue) in place of a negative or neutral charge. In protein nanopores, positive charges may be introduced in the form of positively charged amino acid residues, such as arginine and lysine. Negatively charged amino acid residues and/or neutral amino acid residues may be mutated to positively charged amino acid residues. Alternatively, the amino acid residues of choice may be mutated to cysteine, followed by cysteine reflection An allergic linker (such as maleimide) introduces a positive charge by functionalizing the cysteine residue with a positive charge. In solid state nanopores, the positively charged (or positively charged residues) may be positively charged organic species and/or positively charged inorganic species (e.g., hfO ₂ A coating). In other examples, the positively charged species may be grafted to the surface of the solid state nanopore via an aminosilane or other coupling agent.

Introducing positively charged residues to the inner surface 48 may eliminate or reduce the reactive electrolyte species C ⁺ Transmission through the modified nanopore 20'.

In one example, the modified nanopore 20' is a modified protein in which negatively charged residues, neutral charged residues, or both negatively and neutral charged residues are mutated to positively charged residues. Many protein nanopores exhibit at least 7-fold symmetry, meaning that they consist of seven or more identical polypeptide chains assembled into a loop with symmetry determined by the number of individual chains. This symmetry may allow simultaneous mutation of each subunit of a single type of amino acid residue to completely alter the charge of the inner surface 48. However, in some cases, it may be desirable to mutate less than each subunit of a single type of amino acid residue, thereby introducing less charge into the modified nanopore 20'. In these cases, it may be desirable to mutate some of the subunits of a particular residue (but not all 7, 8, etc.), or by mutating the single strand of the protein nanopore appropriately (e.g., point mutation).

As one example, the protein nanopore is MspA, which is octameric, i.e., has 8-fold symmetry. Positively charged residues may carry a charge of 1 or 2, which enables each mutated residue type to introduce 8 positive charges to 16 positive charges at the inner surface 48 (assuming all 8 residues are mutated). MspA proteins have ultra-narrow (e.g., about 1 nm) and ultra-short (about 2 nm) channels, thus adding about 2.8 positive charges to substantially reduce or eliminate reactive electrolyte species C ⁺ Transmission may be desirable (see example 4).

In one illustrationIn an example, negatively charged aspartic acid residues D139 and D118 of MspA nanopores can be mutated to positively charged arginine or lysine. This positioning can help attract the nucleotide sample to the modified nanopore 20' and also repel the reactive electrolyte species C present in the cis trap 12 ⁺ 。

In another example, negatively charged aspartic acid residues D90, D91, and/or D93 of an MspA nanopore may be mutated to positively charged arginine or lysine. These residues are present in the narrow constriction 60 of the MspA nanopore. Thus, in some examples, a plurality of positively charged residues on the inner surface 48 are located at the constriction region 60 of the modified nanopore 20'. As an example, negatively charged aspartic acid residues D90, D91 and/or D93 may be mutated to D90R, D91R and/or D93R, respectively, which will introduce 1 or 2 positive charges for each subunit. In other examples, negatively charged aspartic acid residues D90, D91, and/or D93 may be mutated to D90K, D91K and/or D93K, respectively, or D90H, D91H and/or D93H. Any combination of D90, D91, and/or D93 mutations may also be produced. Residues D90 and D91 are the most exposed to solvents and therefore may be the largest contributor to cation rejection when mutating to include positively charged residues.

Table 1 lists additional examples of protein nanopores and proposed internal surface modifications that, when used as modified nanopores 20' in nanopore sensor device 10, can achieve reactive electrolyte species C ⁺ Rejection. If the proposed positive charge mutation is made on each subunit of the substituted negatively or neutral charged residue, the proposed net charge is equal to the introduced charge. Thus, it may be desirable to mutate some, but not all 7, 8 or 9, subunits of a particular residue, or to mutate single or double strands of a protein nanopore (e.g., point mutations).

TABLE 1

# represents the second constriction region conferred by the CsgF-binding CsgG well

* Representing calculation of only a small fraction of the total constriction area

The CsgG protein nanopores share a narrow constriction region 60 defined by residues N55 and F56, although this constriction occurs in the middle of the larger pore, with the surface defined by the loops of residues 46-61. The measured size of the hole was 1.3nm in diameter and 0.3nm in height. A net charge of 0.4 may be sufficient to achieve this micro channel (1.2 nm ² ) Is repelled by cations in (a). If the CsgF peptide is added to the CsgG nanopore, a second constriction (from cis to trans) is formed approximately 3nm below the first constriction. The constriction region is defined by residue N17. In this example, positive amino acid mutations disclosed herein can be used to confer ion selectivity at one or both of the constriction regions.

The fragaceae toxin C nanopore is unique in the ssDNA threading nanopore due to the alpha-helical nature of the transmembrane portion, which also defines a narrow constriction region 60, where residue D10 imparts a negative charge. Cation rejection may be achieved by mutating one or more of the eight D10 negatively charged residues.

Aerolysin and alpha-hemolysin (aHL) each have a long and narrow barrel constriction 60, defined by two beta-strands by symmetry. The internal surface charge of the barrel can be adjusted to impart a net positive charge at any point throughout the process. Even with 9.3nm high barrel aerolysin pores, the total net charge of > +11 can be achieved with two modest mutations (D216N and E254Q) that neutralize the two negatively charged side chains on the barrel interior surface and effectively cover the barrel with two positive rings made of R282 and K242. The alpha-hemolysin barrel is small, about 5.5nm in height, but can be similarly tuned to have positively charged rings at the inlet and outlet by neutralizing mutations E111Q and D127N. In this example, the plus ring may be defined by residues K147 and K131.

In another oneIn an example, the modified nanopore 20' is a solid state nanopore in which a positively charged organic species, a positively charged inorganic species, or both a positively charged organic species and a positively charged inorganic species act as positively charged residues. In one example, the positively charged inorganic substance is HfO on a solid state nanopore ₂ And (3) coating. In another example, an aminosilane (e.g., (3-aminopropyl) triethoxysilane, (3-aminopropyl) trimethoxysilane, (3-ethoxydimethylsilyl) propylamine, bis [ 3-trimethoxysilyl) propyl]Amine, etc.) or lysine to the surface to introduce a positive charge.

Examples of methods for detecting ion current to analyze biological compounds include: providing a nanopore 20' within the membrane 18 separating the cis 12 and trans 16 wells, the nanopore 20' having a plurality of positively charged residues on an inner surface 48 of the nanopore 20 '; electrolytes 24 or 24A and 24B are provided within the cis well 12 and trans well 16 (respectively); and applying an electrical current between the cis cathode 14 at least partially exposed to the cis trap 12 and the trans anode 22 at least partially exposed to the trans trap 16 to generate an ionic current through the nanopore 20', wherein a plurality of positively charged residues of the nanopore 20' inhibit the reactive electrolyte species C ⁺ Translocation from the trans well 16 to the cis well 12 occurs during application of an electrical current.

In some examples of the method, the electrolyte solution 24 may include a catalyst capable of dissociating into counter ions (cation C ⁺ And its associated anion A ^- ) Any of the reactive electrolyte species C of (2) ⁺ 、A ^- Wherein one of these counter ions (e.g., cation C ⁺ Or anions A ^- ) Participating in the half-reaction at the cis electrode 14 and the trans electrode 22. The electrolyte solution 24 also includes a polar solvent, such as water.

In other examples of the method, the cis-trap 12 includes an electrolyte solution 24A (having a higher concentration of a reactive electrolyte species C ⁺ 、A ^- ) And trans trap 16 includes electrolyte solution 24B (having a lower concentration of reactive electrolyte species C ⁺ 、A ^- ). Thus, during application of current, cis-well 12 includes a higher level of charge than trans-well 16High concentration of reactive electrolyte species C ⁺ 、A ^- . When the modified nanopore 20' and electrolyte solutions 24A, 24B are used together in the nanopore sensor device 10, several benefits may be realized: the size of the fixed charge of the modified nanopore can be reduced (as opposed to using the same electrolyte solution 24 in the cis well 12 and trans well 16); the magnitude of the concentration gradient may be reduced (as opposed to when unmodified nanopores 20 are used), which reduces the osmotic pressure differential across membrane 18; and the nanopore current may be increased.

When the electrolyte 24 or the respective electrolyte solutions 24A, 24B are contained within the cis-well 12 and trans-well 16, a nucleotide sample may be added to the cis-well 12, for example, via fluid inlets or outlets 56 and 58. The controller 50 may then be used to activate the stimulus source 52 to individually/selectively address one of the plurality of counter electrodes 22. A stimulus source 52 (e.g., current source, voltage source) causes an ionic current to flow through the modified nanopore 20' connected to the addressed counter electrode 22.

In this exemplary method, the application of current between the cis cathode 14 and the trans anode 22 may be initiated by a stimulus 52 that applies a voltage bias between the cis well 12 and at least one of the plurality of trans wells 16 and across the membrane 18. In some examples, the applied current is a unipolar current. In other examples, the applied current is an alternating current between the cis electrode 14 and the addressed trans electrode 22 of the addressed one of the plurality of trans wells 16. In this example, a voltage polarity is typically applied such that negatively charged nucleic acids are driven into the modified nanopore 20' by electrophoresis. By way of example, the voltage is in the range of about 5mV to about 500mV (either polarity).

The cis-trans voltage bias causes ionic current to flow through the modified nanopore 20' associated with the addressed trans anode 22. The current through the modified nanopore 20' forces the corresponding nucleotide with the charge-carrying anion a ^- Together translocate through the modified nanopore 20. The positively charged residues of the modified nanopore 20' can also help attract negatively charged nucleotides. In addition, positively chargedThe charged residues are also capable of rejecting cations C of electrolyte solution 24 or 24A and 24B ⁺ . The ionic current comprises electrolyte C due to cation repulsion caused by the at least partially positively charged surface of the modified nanopore 20 ⁺ 、A ^- A quantity of anions a that translocate through the modified nanopore 20' to the addressed trans trap 16 ^- The A is ^- Is higher than electrolyte C ⁺ 、A ^- Is translocated from the addressed trans trap 16 through the modified nanopore 20 ⁺ Is a combination of the amounts of (a) and (b). This effect may be enhanced when different concentrations of electrolyte solutions 24A, 24B are used in combination with the modified nanopore 20'.

Modified nanopores 20' reduce or block cations C via drift or diffusion ⁺ Transmission through the nanopore 20'. Thus, the initial cation C at t=0 ⁺ The current is reduced or even eliminated. In this way, starting from t=0, anion a ^- Is forced to carry most of the ion current, thus alleviating the initial drop in current and the total current will remain more stable throughout the sensing run. The lifetime of the nanopore sensor device 10 may be extended, in part, because a greater proportion of the current is forced through the nanopore 20 to be additionally restricted by the anions a ^- Is carried by the patient.

Redox inactive buffers

Referring now to fig. 4, one example of a nanopore sensor device 10 includes: an electrolyte solution 24C (in each of the cis 12 and trans 16 traps) comprising a redox inactive buffer 62 comprising a redox inactive species (e.g., cation X) having a diameter greater than the diameter of the constriction region 60 of the nanopore 20 or 20 ⁺ Anions Y ^- ) The method comprises the steps of carrying out a first treatment on the surface of the And redox pairs (e.g., RC ⁺ 、RC ^- ). In these examples, the electrodes 14, 22 are selected to be inactive materials that can transfer electrons rather than exchanging ions.

In these examples, electrolyte solution 24C includes a redox pair RC ⁺ 、RC ^- . By means of redox couples, phasesThe same reactant RC may undergo reduction and oxidation, and the oxidized form (RC ⁺ ) And reduced form (RC) ^- ) Both of which may be present in the electrolyte solution. Any redox pair RC that can transfer electrons can be used ⁺ 、RC ^- . Examples of suitable redox couples include FeCN (e.g.,). Since the FeCN redox couples are all negatively charged, a counter ion is included to maintain the overall charge neutrality of solution 24C. Examples of suitable counterions that can be present include Na ⁺ 、Li ⁺ 、Ca ²⁺ 、K ⁺ . Electrolyte solution 24C also includes a polar solvent, such as water.

When the redox couple is positively charged, cation X of redox inactive buffer 62 is selected ⁺ Such that its diameter is greater than the diameter of the nanopore constriction 60. The bulky redox inactive cation X ⁺ Will be too large to be transported through the nanopore 20 and therefore will not adversely affect the balance of the positively charged redox couple.

Bulky redox inactive cation X ⁺ Comprising a cationic complexing agent and a cation complexed at the center of the cationic complexing agent. The diameter of the central cavity of the cation complexing agent is selected to match the size of the cation. By "matching" is meant that the cation can fit within the central cavity and the atoms of the cation complexing agent can complex with the cation.

Examples of suitable cationic complexing agents include crown ethers, calixarenes, and valinomycin. Examples of suitable crown ethers include:

and +.>It will be appreciated that derivatives of these crown ethers may also be used, such as benzo or dibenzo-15-crown-5, benzo or dibenzo-18-crown-6, benzo or dibenzo-21-crown-7, bicyclicHexao-18-crown-6, dicyclohexyl-21-crown-7, and the like. Aza crown ethers (e.g., aza-15-crown-5) or crown sulfide may also be used. Examples of suitable calixarenes include C ₃ Cal-5、C ₃ Cal-6 and cup [4 ]]Aromatic tetraesters. Valinomycin is shown below:

examples of matching cations and cation complexing agents are shown in table 2.

TABLE 2

The amount of cationic complexing agent may vary depending, at least in part, on the affinity of the complexing agent for the cations in redox inactive buffer 62. In view of this affinity, the cationic complexing agent may be used in any suitable amount that will result in at least 99% of the available cations being complexed to produce a bulky redox inactive cation X ⁺ . Generally, the molar concentration of the cationic complexing agent can range from greater than 0mM to about 1M. In an example, 18-crown-6 can be used in a potassium-containing electrolyte at a molar concentration in the range of about 50mM to about 500 mM. In a more specific example, 18-crown-6 can be used in a potassium-containing electrolyte at a molar concentration of about 300 mM. In another example, calixarene can be used in potassium-containing or sodium-containing electrolytes at molar concentrations in the range of greater than 0mM to about 20 mM. Any of the concentrations used may depend on the solubility of the cationic complexing agent in the electrolyte solution 24C.

When the redox couple is positively charged, the anion Y of the redox inactive buffer 62 ^- May be any anion that is redox inactive at the electrodes 14, 22 of the nanopore sensor device 10. Any anion Y can be used ^- Because the electrodes 14, 22 are inactive. In an example, the anion Y ^- Is chloride ion.

When the redox couple is negatively charged, the anion Y of the redox inactive buffer 62 is selected ^- Such that its diameter is greater than the diameter of the nanopore constriction 60. The bulky redox inactive anion Y ^- Will be too large to be transported through the nanopore 20 or modified nanopore 20' and therefore will not adversely affect the balance of the negatively charged redox species.

When the redox species is negatively charged, cation X of redox inactive buffer 62 ⁺ May be any cation that is redox inactive at the electrodes 14, 22 of the nanopore sensor device 10. Any cation C can be used ⁺ Because the electrodes 14, 22 are inactive. Examples of suitable cations that may be present include Na ⁺ 、Li ⁺ 、Ca ²⁺ 、K ⁺ 。

When electrolyte 24C is contained within cis-well 12 and trans-well 16, a nucleotide sample may be added to cis-well 12, for example, via fluid inlets or outlets 56 and 58. The controller 50 may then be used to activate the stimulus 52 to individually/selectively address one of the plurality of trans-anodes 22. A stimulus source 52 (e.g., current source, voltage source) causes ionic current to flow through the nanopore 20 or modified nanopore 20' connected to the addressed trans anode 22.

In one example, stimulus 52 applies a voltage bias (using electrodes 14, 22) between cis-well 12 and at least one of the plurality of trans-wells 16 and thus across membrane 18. The magnitude and polarity (positive or negative) of the applied voltage bias may depend in part on the concentration of the redox couple in electrolyte solution 24C. The size and polarity can be adjusted to account for the diffusivity and mobility of the redox couple. In an example, the voltage bias between the cis well 12 and the addressed electrode 22 in one of the plurality of trans wells 16 is in the range of about-1V to about 1V. Any voltage bias within a given range may be applied when the electrolyte solution 24C is present in the respective well 12, 16.

In some examples, the controller 50 is configured to cause the stimulus 52 to apply a unipolar current between the cis electrode 14 and the addressed trans electrode 22 of the addressed transwell 16 of the plurality of transwells 16. As one example, when the redox couple is negatively charged and thus is the desired species to carry charge, a positive current is induced between the wells 12, 16 and thus through the nanopore 20 or 20' and membrane 18. As another example, when the redox couple is positively charged and thus is the desired species to carry a charge, a negative current is induced between the wells 12, 16 and thus through the nanopore 20 or 20' and membrane 18. In other examples, the controller 50 is configured to cause the stimulus 52 to apply an alternating current between the cis electrode 14 and the addressed trans electrode 22 of the addressed one of the plurality of trans traps 16.

The cis-trans voltage bias causes an ion current to flow through the nanopore 20 or 20' associated with the addressed trans electrode 22. The current through the nanopore 20 or 20 'forces the corresponding nucleotide to translocate through the modified nanopore 20 or 20' along with the negatively charged redox pair carrying the charge. The bulk ion of the non-active redox buffer 62 is too large to translocate the nanopore 20 or 20'.

When modified nanopore 20 'is used in this example, the positively charged residues of modified nanopore 20' may help attract negatively charged nucleotides, as well as repel cation C of redox inactive buffer 62 ⁺ And cations used in conjunction with redox pairs. Due to the cation repulsion caused by the at least partially positively charged surface of the modified nanopore 20', the ion current includes an amount of anionic RC in the redox pair that translocates through the modified nanopore 20' to the addressed trans well 16 ^- The RC is provided with ^- Is higher than the amount of anion a translocated from the addressed trans trap 16 through the modified nanopore 20' in the redox inactive buffer 62 ^- Is a combination of the amounts of (a) and (b).

For further explanation of the present disclosure, examples are set forth herein. It should be understood that the examples are provided for illustrative purposes and are not to be construed as limiting the scope of the present disclosure.

Non-limiting working examples

Comparative example 1

In this comparative example, it is calculated that the same K is present ⁺ Cl ^- Concentration nanopore sensor behavior in cis-well and trans-well. The time and material dependence of the nanopore current was obtained by numerically solving the Nernst-Planck equation in a commercial software package (Comsol).

Calculation was performed for a nanopore sensor with the following: cis-well with diameter of 200 μm and height of 200 μm, trans-well with diameter of 20 μm and height of 20 μm, K ⁺ Cl ^- Each well at a concentration of 100mM and a cis-trans bias voltage of about 200 mV.

Fig. 5A shows the time dependence of the current, and fig. 5B shows the material decomposition of the current components.

From this simulation, it is evident that the current decays rapidly in about 50 minutes, which is consistent with the calculation of equation (1) (this calculation is performed using the parameters set forth in this example and using chloride as the reactive species). As shown in fig. 5B, at time t=0, the current is represented by K ⁺ Cations and Cl ^- The anions are carried in equal amounts. Over time, both components drop, the cationic component drops much more rapidly, reaching a point where the current is almost carried by the anions only. At this time, cl ^- The supply and consumption of anions reach equilibrium and a new equilibrium is reached. However, this new equilibrium is at a much lower current level (e.g., about 15% of the initial current) that cannot actually be used for the nanopore sensor device.

Example 2

In this example, K in the cis trap was calculated ⁺ Cl ^- Concentration is higher than K in trans-well ⁺ Cl ^- Behavior of the concentration nanopore sensor. The time and material dependence of the nanopore current was obtained by numerically solving the Nernst-Planck equation in a commercial software package (Comsol).

Calculation was performed for a nanopore sensor with the following: cis-well, K, with a diameter of 200 μm and a height of 200 μm ⁺ Cl ^- Cis-trap at 500mM concentrationTrans-well, K, with diameter of 20 μm and height of 20 μm ⁺ Cl ^- A trans trap at a concentration of 100mM and a cis-trans bias voltage of about 80 mV.

Fig. 6A shows the time dependence of the current, and fig. 6B shows the material decomposition of the current components.

As shown in fig. 6B, at time t=0, since the current portion of the chloride anion is 10 times greater than that of the potassium cation, the equilibrium condition (about 60% of the initial current) that is actually useful is different from the comparative example (having equal electrolyte concentrations in the cis and trans traps).

Example 3

In this example, K in the cis trap was calculated ⁺ Cl ^- Concentration is higher than K in trans-well ⁺ Cl ^- Behavior of another nanopore sensor of concentration. The time and material dependence of the nanopore current was obtained by numerically solving the Nernst-Planck equation in a commercial software package (Comsol).

Calculation was performed for a nanopore sensor with the following: cis-well, K, with a diameter of 200 μm and a height of 200 μm ⁺ Cl ^- Cis-well with a concentration of 850mM, trans-well with a diameter of 20 μm and a height of 20 μm, K ⁺ Cl ^- A trans trap at a concentration of 100mM and a cis-trans bias voltage of about 55.35 mV.

Fig. 7A shows the time dependence of the current, and fig. 7B shows the material decomposition of the current component.

As shown in FIG. 7B, K ⁺ Transmission ratio Cl in the whole pore ^- The component is approximately 3 orders of magnitude lower, therefore Cl ^- Is balanced and the hole current is stable. In this example, the concentration gradient and cis-trans bias completely offset the drift and diffusion cation currents, but the total current is approximately equal to that of comparative example 1.

Example 4

To assess whether ultra-narrow and ultra-short channels of biological nanopores are capable of cation exclusion at desired levels, 3D finite element analysis of geometry was performed, as shown in fig. 8. The complete structure is rotationally symmetrical, so only half of the analog domain is shown in fig. 8.

In this analysis, the nanopores were 1nm wide and 2nm high, which approximates the size of the MspA constriction. Different amounts of fixed surface charge are placed on the surface of the constriction. Placing the "cis" well and the "trans" well in analog K ⁺ And Cl ^- To eliminate depletion effects from the simulation.

For 0.01q/nm ² To 1q/nm ² The surface charge density in the range was simulated, where q is the base charge. This is the net charge that would exist in an idealized "anhydrous" state in the absence of electrolyte. In principle, such charge densities are achievable in protein nanopores.

For sigma=0.01 q/nm ² 、σ＝0.1q/nm ² Sigma=1q/nm ² The results of the simulation are shown in fig. 9A, 9B, and 9C, respectively. More specifically, FIG. 9 shows the calculated spatial distribution (logarithmic scale) of the Cl to K ratio in the analog domain. The data in fig. 9A, 9B and 9C are plotted on a logarithmic scale, and each image is accompanied by scale markings. Although the Cl to K ratio peaks in the nanochannel for all three conditions, the magnitude of the effect is exponentially related to the charge density. This is more clearly visible in fig. 10. FIG. 10 is a calculated correlation of Cl to K flux ratio through a nanopore and magnitude of fixed charge density in the pore channel.

To characterize the inhibition of total K through nanopores ⁺ Flux benefit as in example 1 (d=200 μm/h=200 μm cis-well; d=20 μm/h=20 μm trans-well; 100mM K in cis-well and trans-well) ⁺ Cl; and 100mV for a cis-trans bias) additional simulations were performed at three different Cl: K flux ratio values: r=1.2 (σ=0.01 q/nm) ² )；R＝5(σ＝0.1q/nm ² ) The method comprises the steps of carrying out a first treatment on the surface of the And r=40 (σ=0.4 q/nm ² ). The results are shown in fig. 11A, 11B, and 11C, respectively. From these results, it was found that K was suppressed ⁺ The beneficial effects of transport through the nanopore are apparent. At r=40 (σ=0.4 q/nm ² ) The depletion effect is almost completely suppressed. The current drift is significantly reduced, which is advantageous for practical use of the nanopore sensor.

Example 5

In this example, four different types of modified MspA wells were compared. The comparative MspA well includes neutral asparagine residues at D90, D91 and D93. The first example MspA well includes neutral asparagine residues at D90 and D93 and a positively charged arginine residue at D91. The second example MspA well includes neutral asparagine residues at D91 and D93 and positively charged arginine residues at D90. The third example MspA well includes a neutral asparagine residue at D93 and positively charged arginine residues at D90 and D91. All wells were tested using wells 5 μm deep and 16 μm wide. The data shown in this example represent the average results for ten or more corresponding wells (e.g., 19 comparative MspA wells tested at 150mM, 58 first example MspA wells tested at 150mM, 21 comparative MspA wells tested at 300mM, and 10 first example MspA wells tested at 300 mM).

In the first experiment, K in cis-well and trans-well ⁺ Cl ^- The concentration was 300mM. The cis-trans voltage bias was swept from-175 mV to 175mV and the current (nA) was recorded. Fig. 12A shows a graph of current (nA, Y-axis) versus voltage (mV, X-axis) for the comparative MspA well, and fig. 12B shows a graph of current versus voltage for the first embodiment MspA well. Although the data for the second and third example MspA wells are not reproduced herein, all wells (comparative and example MspA wells) exhibit higher resistance at higher positive potentials. The first and second example MspA wells (each with two neutral residues) exhibited the expected current rectification under negative bias (see fig. 12B for the results of the first example MspA well). The third example MspA well (with two positive residues) has little conductivity before using a large positive potential.

In a second experiment, a different K was used ⁺ Cl ^- Concentration (i.e., 150mM and 300 mM) of the test comparative example MspA wells and the first example MspA wells. Will be biasedThe voltage was swept from 50mV to 150mV and the current (pA) was recorded. Fig. 13 plots average current (pA, Y axis) versus cis-trans voltage bias (mV, X axis) in the first example well and the comparative example well. It is apparent that the first example MspA well has higher conductivity than the comparative example MspA well, regardless of electrolyte concentration.

In a third experiment, 150mM K was used ⁺ Cl ^- The comparative and first example MspA wells were tested for concentration and cis-trans bias voltage of 50 mV. Fig. 14A plots current (pA, Y axis) versus time (s, X axis) for the comparative MspA well, and fig. 14B plots extrapolated current (pA, Y axis) versus time (s, X axis) for the first example MspA well. The comparative example wells showed significant current decay over the first 20 minutes, whereas the first example MspA wells showed no current decay.

Additional description

It is to be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (assuming such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be understood that terms explicitly employed herein, which may also appear in any disclosure incorporated by reference, should be given the most consistent meaning with the particular concepts disclosed herein.

Reference throughout this specification to "one example," "another example," "an example," etc., means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. Furthermore, it should be understood that the elements described for any example may be combined in any suitable manner in the various examples unless the context clearly indicates otherwise.

Although a few examples have been described in detail, it should be understood that modifications can be made to the disclosed examples. Accordingly, the above description should be regarded as non-limiting.

Claims

1. A nanopore sensor device, comprising:

one or more cis traps;

a cis electrode;

a plurality of trans traps, each trans trap of the plurality of trans traps separated from the one or more cis traps by a lipid/polymer/solid state membrane having nanopores;

a plurality of counter electrodes, each of the plurality of counter electrodes being associated with one of the plurality of counter wells;

a first concentration of electrolyte, the first concentration of electrolyte being located within the one or more cis wells; and

a second concentration of the electrolyte, the second concentration of the electrolyte being located within the trans trap, wherein the first concentration is higher than the second concentration.

2. The nanopore sensor device of claim 1, further comprising:

a stimulus source coupled to each counter electrode of the plurality of counter electrodes, either alone or via multiplexing, wherein the stimulus source will flow an electrical current through the nanopore; and

A controller coupled to the stimulus source, the controller configured to individually/selectively address the plurality of counter electrodes to pass ion current through the nanopores of the addressed counter electrodes of the plurality of counter wells.

3. The nanopore sensor device of claim 2, wherein the ionic current comprises an amount of anions in the electrolyte that translocate through the nanopore to an addressed trans well, the amount of anions being greater than an amount of cations in the electrolyte that translocate from the addressed trans well through the nanopore.

4. The nanopore sensor device of claim 2, wherein the controller is further configured to cause the stimulus to apply a unipolar current between the cis electrode and the addressed trans electrode of the addressed trans well of the plurality of trans wells.

5. The nanopore sensor device of claim 1, wherein the nanopore has a plurality of positively charged residues on an inner surface of the nanopore.

6. The nanopore sensor device of claim 5, wherein the plurality of positively charged residues on the inner surface are located at a constriction region of the nanopore.

7. The nanopore sensor device of claim 1, wherein the ratio of the first concentration to the second concentration is in the range of about 10:1 to about 3:1.

8. A nanopore sensor kit comprising:

a nanopore sensor device, the nanopore sensor device comprising:

one or more cis traps, the one or more cis traps comprising a fluid inlet;

a cis electrode;

a plurality of counter electrodes, each of the plurality of counter electrodes being associated with one of the plurality of counter wells; and

a first concentration of an electrolyte, the first concentration of the electrolyte being located within the one or more cis-wells and the plurality of trans-wells; and

a second concentration of the electrolyte to be introduced into the one or more cis-traps through the fluid inlet upon initial cycling of the nanopore sensor device such that the one or more cis-traps contain the second concentration of the electrolyte and the plurality of trans-traps contain the first concentration of the electrolyte, wherein the second concentration is higher than the first concentration.

9. A method of detecting ion current for analysis of biological compounds, comprising:

providing a nanopore within a membrane separating a cis-well and a trans-well, the nanopore having a plurality of positively charged residues on an inner surface;

providing an electrolyte within the cis well and the trans well; and

applying a current between a cis cathode at least partially exposed to the cis trap and a trans anode at least partially exposed to the trans trap to generate an ionic current through the nanopore,

wherein the plurality of positively charged residues of the nanopore inhibit translocation of cations from the trans well to the cis well during application of the current.

10. The method of claim 9, wherein the plurality of positively charged residues on the inner surface are located at a constriction region of the nanopore.

11. The method of claim 9, wherein the nanopore is a modified protein in which negatively charged residues, neutral charged residues, or both negatively and neutral charged residues are mutated to the positively charged residues.

12. The method of claim 9, wherein the nanopore is a solid state nanopore, wherein a positively charged organic species, a positively charged inorganic species, or both a positively charged organic species and a positively charged inorganic species are the positively charged residue.

13. The method of claim 9, wherein the applied current is a unipolar current.

14. The method of claim 9, wherein during application of the current, the cis well comprises a higher concentration of the electrolyte than the trans well.

15. The method of claim 9, wherein one of:

the electrolyte is a redox pair having a negative charge and is incorporated into a redox inactive buffer comprising anions having a diameter greater than the diameter of the constriction region of the nanopore; and

the electrolyte is a redox pair having a positive charge and is incorporated into a redox inactive buffer comprising cations having a diameter greater than the diameter of the constriction region of the nanopore.

16. A nanopore sensor device, comprising:

one or more cis traps;

a cis electrode;

An electrolyte solution, the electrolyte solution comprising:

a redox inactive buffer comprising a redox inactive species having a diameter greater than a diameter of a constriction region of the nanopore; and

a redox couple.

17. The nanopore sensor device of claim 16, further comprising:

18. The nanopore sensor device of claim 17, wherein the ionic current comprises an amount of the redox couple translocated to the addressed counter electrode through the nanopore, but not an amount of the redox inactive species.

19. The nanopore sensor device of claim 17, wherein the controller is further configured to apply a unipolar current between the cis electrode and the addressed trans electrode of the addressed one of the plurality of trans wells.

20. The nanopore sensor device of claim 16, wherein the nanopore has a plurality of positively charged residues on an inner surface of the nanopore.

21. A nanopore sensor device, comprising:

one or more cis traps;

a cis electrode;

a plurality of trans-traps, each of the plurality of trans-traps separated from the one or more cis-traps by a lipid/polymer/solid state membrane having a nanopore with a plurality of positively charged residues on an inner surface of the nanopore; and

a plurality of counter electrodes, each of the plurality of counter electrodes being associated with one of the plurality of counter wells.